原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因
Summary
Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.
Abstract
The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.
Introduction
菌发挥各种口腔疾病如牙周炎，牙髓炎，冠周炎，蜂窝组织炎，骨髓炎的病因中起作用。为了了解细菌在疾病的发病机制中的作用，并监测治疗的效果，在组织内的定位的细菌是重要的。细菌牙龈组织内与牙周炎的存在从患者已经使用各种方法被证明，包括使用荧光-电子显微镜1,2，免疫组织化学和免疫荧光细菌特异性抗体3-7，和荧光原位杂交（FISH）8标记的寡核苷酸探针靶向16S rRNA基因。然而，这些方法都没有得到广泛由于技术限制或困难使用。与抗体相比，靶向16S rRNA基因的探针是容易产生和实现的种特异性。鱼已被证明是一个极好的工具BACT的可视化ERIA在其天然环境中，如菌斑生物膜。然而，应用FISH对组织样品是由于各种组织成分的自体荧光的限制。例如，红血细胞的强的自体荧光常常妨碍荧光技术对发炎组织的应用程序时，它们涉及出血9。
为了在发炎的牙龈组织内细菌定位，因此，使用地高辛原位杂交法（DIG）标记的DNA探针已经开发并成功应用10,11。这里细菌利用P.石蜡包埋组织内的本地化的详细协议牙龈特异性和通用的真细菌的探针进行了说明。它特别着重于方法，使得类似的结果可以在其他实验室被再现的标准化。该协议允许他们组织上下文和RESU内细菌的定位LTS是高度可重复的。所描述的协议可以用于无论是在各种组织中的一种特异性或通用方式细菌本地化。的通用探针是特别有用的，以检测多微生物疾病的细菌，并研究细菌的潜在作用，在疾病，其中具体的细菌的作用是未知的。
Protocol
Representative Results
Discussion
这里的协议来定位使用已经描述了DIG标记的DNA探针石蜡包埋组织中的细菌。探针靶向细菌16S rRNA基因的DNA或RNA分子，和16S rRNA基因靶向探针可设计为任意的种特异性或通用。在P的特异性杂交牙龈特异性探针P.牙龈而不是其他口腔内细菌先前已经证明10。与此相反，在真细菌探针杂交至所有细菌的基因组DNA进行测试（17种不同的种），尽管杂交效率变化12。的探针?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究是由来自韩国国家研究基金会的资助（2013R1A1A3005669）和韩国保健技术研发项目，卫生部与福利的补助金（HI13C0016）的支持。
Materials
| Acetic anhydride | Sigma | 6404 | |
| 50% Dextran sulfate solution | Millipore | S4030 | |
| 50X Denhardt’s solution | Sigma | D2532 | |
| DEPC | Sigma | P159220 | |
| DIG DNA labeling and detection kit | Roche | 11 093 657 910 | |
| Formamide | Sigma | F9037 | |
| ImmEdge™ Pen | Dako | H-400 | |
| Levamisole | Vector | SP-5000 | |
| Magnesium chloride | Sigma | 246964 | |
| Maleic acid | Sigma | M0375 | |
| Methyl green | Sigma | M6776 | |
| Paraformaldehyde | Sigma | P1648 | |
| Permount | Fisher | SP15-500 | |
| Salmon sperm DNA solution | Invitrogen | #15632-011 | |
| Sodium chloride | Sigma | S9625 | |
| Sodium citrate | Duksan | D1420 | |
| Sodium dodecyl sulfate | Amresco | 227 | |
| Triethanolamine-HCl | Sigma | 90279 | |
| Tris-HCl | Research organics | 3098T |
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